Soybean cultivar 9469069618964

ABSTRACT

A novel soybean cultivar, designated 9469069618964, is disclosed. The invention relates to the seeds of soybean cultivar 9469069618964, to the plants of soybean 9469069618964 and to methods for producing a soybean plant produced by crossing the cultivar 9469069618964 with itself or another soybean variety. The invention further relates to hybrid soybean seeds and plants produced by crossing the cultivar 9469069618964 with another soybean cultivar.

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinctive soybean cultivar,designated 9469069618964. There are numerous steps in the development ofany novel, desirable plant germplasm. Plant breeding begins with theanalysis and definition of problems and weaknesses of the currentgermplasm, the establishment of program goals, and the definition ofspecific breeding objectives. The next step is selection of germplasmthat possess the traits to meet the program goals. The goal is tocombine in a single variety an improved combination of desirable traitsfrom the parental germplasm. These important traits may include higherseed yield, resistance to diseases and insects, better stems and roots,tolerance to drought and heat, and better agronomic quality.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination, and thenumber of hybrid offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for three or more years. The best lines are candidatesfor new commercial cultivars; those still deficient in a few traits maybe used as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from eight to 12 years from the time thefirst cross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of plant breeding is to develop new, unique and superiorsoybean cultivars and hybrids. The breeder initially selects and crossestwo or more parental lines, followed by repeated selfing and selection,producing many new genetic combinations. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,selfing and mutations. The breeder has no direct control at the cellularlevel. Therefore, two breeders will never develop the same line, or evenvery similar lines, having the same soybean traits.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The cultivarswhich are developed are unpredictable. This unpredictability is becausethe breeder's selection occurs in unique environments, with no controlat the DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce the same cultivar twice by using theexact same original parents and the same selection techniques. Thisunpredictability results in the expenditure of large amounts of researchmonies to develop superior new soybean cultivars.

The development of new soybean cultivars requires the development andselection of soybean varieties, the crossing of these varieties andselection of superior hybrid crosses. The hybrid seed is produced bymanual crosses between selected male-fertile parents or by using malesterility systems. These hybrids are selected for certain single genetraits such as pod color, flower color, pubescence color or herbicideresistance which indicate that the seed is truly a hybrid. Additionaldata on parental lines, as well as the phenotype of the hybrid,influence the breeder's decision whether to continue with the specifichybrid cross.

Pedigree breeding and recurrent selection breeding methods are used todevelop cultivars from breeding populations. Breeding programs combinedesirable traits from two or more cultivars or various broad-basedsources into breeding pools from which cultivars are developed byselfing and selection of desired phenotypes. The new cultivars areevaluated to determine which have commercial potential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops. Two parents which possess favorable,complementary traits are crossed to produce an F₁. An F₂ population isproduced by selfing one or several F₁'s. Selection of the bestindividuals may begin in the F₂ population; then, beginning in the F₃,the best individuals in the best families are selected. Replicatedtesting of families can begin in the F₄ generation to improve theeffectiveness of selection for traits with low heritability. At anadvanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting plant is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, soybean breeders commonly harvest one ormore pods from each plant in a population and thresh them together toform a bulk. Part of the bulk is used to plant the next generation andpart is put in reserve. The procedure has been referred to as modifiedsingle-seed descent or the pod-bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster to thresh pods with a machine than to remove oneseed from each by hand for the single-seed procedure. The multiple-seedprocedure also makes it possible to plant the same number of seeds of apopulation each generation of inbreeding. Enough seeds are harvested tomake up for those plants that did not germinate or produce seed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr,1987).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer; for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Soybean, Glycine max (L), is an important and valuable field crop. Thus,a continuing goal of plant breeders is to develop stable, high yieldingsoybean cultivars that are agronomically sound. The reasons for thisgoal are obviously to maximize the amount of grain produced on the landused and to supply food for both animals and humans. To accomplish thisgoal, the soybean breeder must select and develop soybean plants thathave the traits that result in superior cultivars.

SUMMARY OF THE INVENTION

According to the invention, there is provided a novel soybean cultivar,designated 9469069618964. This invention thus relates to the seeds ofsoybean cultivar 9469069618964, to the plants of soybean 9469069618964and to methods for producing a soybean plant produced by crossing thesoybean 9469069618964 with itself or another soybean line, and thecreation of variants by mutagenesis or transformation of soybean9469069618964.

Thus, any such methods using the soybean variety 9469069618964 are partof this invention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using soybean variety9469069618964 as a parent are within the scope of this invention.Advantageously, the soybean variety could be used in crosses with other,different, soybean plants to produce first generation (F₁) soybeanhybrid seeds and plants with superior characteristics.

In another aspect, the present invention provides for single or multiplegene converted plants of 9469069618964. The transferred gene(s) maypreferably be a dominant or recessive allele. Preferably, thetransferred gene(s) will confer such traits as herbicide resistance,insect resistance, resistance for bacterial, fungal, or viral disease,male fertility, male sterility, enhanced nutritional quality, andindustrial usage. The gene may be a naturally occurring soybean gene ora transgene introduced through genetic engineering techniques.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of soybean plant 9469069618964. The tissue culturewill preferably be capable of regenerating plants having thephysiological and morphological characteristics of the foregoing soybeanplant, and of regenerating plants having substantially the same genotypeas the foregoing soybean plant. Preferably, the regenerable cells insuch tissue cultures will be embryos, protoplasts, meristematic cells,callus, pollen, leaves, anthers, roots, root tips, flowers, seeds, podsor stems. Still further, the present invention provides soybean plantsregenerated from the tissue cultures of the invention.

Definitions

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. Allele is any of one or more alternative forms of a gene, all ofwhich alleles relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotypes of the F₁hybrid.

Brown Stem Rot. This is a visual disease score from 1 to 9 comparing allgenotypes in a given test. The score is based on leaf symptoms ofyellowing and necrosis caused by brown stem rot. A score of 1 indicatesno symptoms. Visual scores range to a score of 9 which indicates severesymptoms of leaf yellowing and necrosis.

Cotyledon. A cotyledon is a type of seed leaf. The cotyledon containsthe food storage tissues of the seed.

Embryo. The embryo is the small plant contained within a mature seed.

Emergence. This score indicates the ability of the seed to emerge whenplanted 3″ deep in sand and with a controlled temperature of 25° C. Thenumber of plants that emerge each day are counted. Based on this data,each genotype is given a 1 to 9 score based on its rate of emergence andpercent of emergence. A score of 1 indicates an excellent rate andpercent of emergence, an intermediate score of 5 indicates averageratings and a 9 score indicates a very poor rate and percent ofemergence.

Hilum. This refers to the scar left on the seed which marks the placewhere the seed was attached to the pod prior to the seed beingharvested.

Hypocotyl. A hypocotyl is the portion of an embryo or seedling betweenthe cotyledons and the root. Therefore, it can be considered atransition zone between shoot and root.

Iron-Deficiency Chlorosis. Plants are scored 1 to 9 based on visualobservations. A score of 1 means no stunting of the plants or yellowingof the leaves and a score of 9 indicates the plants are dead or dyingcaused by iron-deficiency chlorosis, a score of 5 means plants haveintermediate health with some leaf yellowing.

Lodging Resistance. Lodging is rated on a scale of 1 to 9. A score of 1indicates erect plants. A score of 5 indicates plants are leaning at a45° angle in relation to the ground and a score of 9 indicates plantsare laying on the ground.

Maturity Date. Plants are considered mature when 95% of the pods havereached their mature color. The number of days are either calculatedfrom August 31 or from the planting date.

Maturity Group. This refers to an agreed-on industry division of groupsof varieties, based on zones in which they are adapted primarilyaccording to day length or latitude. They consist of very long daylength varieties (Groups 000, 00, 0), and extend to very short daylength varieties (Groups VII, VIII, IX, X).

Oil or oil percent. Soybean seeds contain a considerable amount of oil.Oil is measured by NIR spectrophotometry, and is reported on an as ispercentage basis.

Oleic Acid Percent. Oleic acid is one of the five most abundant fattyacids in soybean seeds. It is measured by gas chromatography and isreported as a percent of the total oil content.

Palmitic Acid Percent. Palmitic acid is one of the five most abundantfatty acids in soybean seeds. It is measured by gas chromatography andis reported as a percent of the total oil content.

Phytophthora Tolerance. Tolerance to Phytophthora root rot is rated on ascale of 1 to 9, with a score of 1 being the best or highest toleranceranging down to a score of 9 which indicates the plants have notolerance to Phytophthora.

Phenotypic Score. The Phenotypic Score is a visual rating of generalappearance of the variety. All visual traits are considered in the scoreincluding healthiness, standability, appearance and freedom of disease.Ratings are scorred from 1 being excellent to 9 being poor.

Plant Height. Plant height is taken from the top of soil to top node ofthe plant and is measured in inches.

Pod. This refers to the fruit of a soybean plant. It consists of thehull or shell (pericarp) and the soybean seeds.

Protein Percent. Soybean seeds contain a considerable amount of protein.Protein is generally measured by NIR spectrophotometry, and is reportedon an as is percentage basis.

Pubescence. This refers to a covering of very fine hairs closelyarranged on the leaves, stems and pods of the soybean plant.

Quantitative Trait Loci (QTL). Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Seed Protein Peroxidase Activity. Seed protein peroxidase activity isdefined as a chemical taxonomic technique to separate cultivars based onthe presence or absence of the peroxidase enzyme in the seed coat. Thereare two types of soybean cultivars, those having high peroxidaseactivity (dark red color) and those having low peroxidase activity (nocolor).

Seed Yield (Bushels/Acre). The yield in bushels/acre is the actual yieldof the grain at harvest.

Seeds per Pound. Soybean seeds vary in seed size, therefore, the numberof seeds required to make up one pound also varies. This affects thepounds of seed required to plant a given area, and can also impact enduses.

Shattering. The amount of pod dehiscence prior to harvest. Poddehiscence involves seeds falling from the pods to the soil. This is avisual score from 1 to 9 comparing all genotypes within a given test. Ascore of 1 means pods have not opened and no seeds have fallen out. Ascore of 5 indicates approximately 50% of the pods have opened, withseeds falling to the ground and a score of 9 indicates 100% of the podsare opened.

Single Gene Converted (Conversion). Single gene converted (conversion)plant refers to plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of a variety are recovered in additionto the single gene transferred into the variety via the backcrossingtechnique or via genetic engineering.

DETAILED DESCRIPTION OF THE INVENTION

9469069618964 is a late maturity group 0 variety with resistance toRoundup™ herbicide conferring tolerance to glyphosate herbicides.9469069618964 has very high yield potential when compared to lines ofsimilar maturity and has excellent agronomic characteristics includinglodging resistance. 9469069618964 has a gene for Phytophthora Root Rotresistance, Rps1a, conferring resistance to several races ofPhytophthora Root Rot.

Some of the criteria used to select in various generations include: seedyield, lodging resistance, emergence, disease tolerance, maturity, lateseason plant intactness, plant height and shattering resistance.

The cultivar has shown uniformity and stability, as described in thefollowing variety description information. It has been self-pollinated asufficient number of generations with careful attention to uniformity ofplant type. The line has been increased with continued observation foruniformity.

Soybean cultivar 9469069618964 has the following morphologic and othercharacteristics (based primarily on data collected at Redwood Falls,Minn.).

Variety Description Information

1. Seed Shape: Spherical Flattened (L/W ratio>1.2; L/T ratio=<1.2)

2. Hilum Color: (Mature Seed)—Yellow

3. Pod color: Brown

4. Seed Coat Luster: Dull

5. Seed Coat Color: Yellow

6. Flower Color: Purple

7. Leaflet Shape: Ovate

8. Leaf Size: Medium

9. Leaf Color: Medium Green

10. Canopy: Intermediate

11. Plant Pubescence Color: Gray

12. Growth Habit: Indeterminate

13. Maturity Group: 0

14. Relative Maturity: 0.9

15. Physiological Responses:

Roundup Ready™ Herbicide: Resistant

16. Disease Resistance:

Phytophthora Rot (Phytophthora megasperma var. sojae): Race 1 ResistantRace 2 Resistant Race 10 Resistant Race 11 Resistant Race 13 ResistantRace 14 Resistant Race 15 Resistant Race 16 Resistant Race 17 ResistantRace 18 Resistant Race 24 Resistant Race 26 Resistant Race 27 ResistantRace 31 Resistant Race 32 Resistant

This invention is also directed to methods for producing a soybean plantby crossing a first parent soybean plant with a second parent soybeanplant, wherein the first or second soybean plant is the soybean plantfrom the line 9469069618964. Further, both first and second parentsoybean plants may be from the cultivar 9469069618964. Therefore, anymethods using the cultivar 9469069618964 are part of this invention:selfing, backcrosses, hybrid breeding, and crosses to populations. Anyplants produced using cultivar 9469069618964 as a parent are within thescope of this invention.

Useful methods include but are not limited to expression vectorsintroduced into plant tissues using a direct gene transfer method suchas microprojectile-mediated delivery, DNA injection, electroporation andthe like. More preferably expression vectors are introduced into planttissues using the microprojectile media delivery with the biolisticdevice Agrobacterium-medicated transformation. Transformant plantsobtained with the protoplasm of the invention are intended to be withinthe scope of this invention.

The cultivar 9469069618964 is similar to A0868. While similar to A0868,there are numerous differences including; 9469069618964 has the gene forresistance to the RoundUp™ herbicides and A0868 does not contain thisgene.

FURTHER EMBODIMENTS OF THE INVENTION

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed variety or line.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used alone or incombination with other plasmids, to provide transformed soybean plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the soybean plant(s).

Expression Vectors for Soybean Transformation

Marker Genes—Expression vectors include at least one genetic marker,operably linked to a regulatory element (a promoter, for example) thatallows transformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) under the control of plantregulatory signals confers resistance to kanamycin. Fraley et al., Proc.Natl. Acad. Sci. U.S.A., 80:4803 (1983). Another commonly usedselectable marker gene is the hygromycin phosphotransferase gene whichconfers resistance to the antibiotic hygromycin. Vanden Elzen et al.,Plant Mol. Biol., 5:299 (1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford et al., Plant Physiol.86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab etal., Plant Mol. Biol. 14:197 (1990<Hille et al., Plant Mol. Biol. 7:171(1986). Other selectable marker genes confer resistance to herbicidessuch as glyphosate, glufosinate or broxynil. Comai et al., Nature317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618 (1990) andStalkeret al., Science 242:419-423 (1988).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67(1987), Shah et al., Science 233:478 (1986), Charest et al., Plant CellRep. 8:643 (1990).

Another class of marker genes for plant transformation require screeningof presumptively transformed plant cells rather than direct geneticselection of transformed cells for resistance to a toxic substance suchas an antibiotic. These genes are particularly useful to quantify orvisualize the spatial pattern of expression of a gene in specifictissues and are frequently referred to as reporter genes because theycan be fused to a gene or gene regulatory sequence for the investigationof gene expression. Commonly used genes for screening presumptivelytransformed cells include β-glucuronidase (GUS, β-galactosidase,luciferase and chloramphenicol, acetyltransferase. Jefferson, R. A.,Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBO J. 8:343 (1989),Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131 (1987), DeBlock etal., EMBO J. 3:1681 (1984).

Recently, in vivo methods for visualizing GUS activity that do notrequire destruction of plant tissue have been made available. MolecularProbes publication 2908, Imagene Green™, p. 1-4 (1993) and Naleway etal., J. Cell Biol. 115:151a (1991). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie et al., Science 263:802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Promoters—Genes included in expression vectors must be driven bynucleotide sequence comprising a regulatory element, for example, apromoter. Several types of promoters are now well known in thetransformation arts, as are other regulatory elements that can be usedalone or in combination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which intitiate transcription only in certain tissue arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression insoybean. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in soybean. With an inducible promoter the rateof transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Mett et al., PNAS 90:4567-4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz etal., Mol. Gen. Genetics 227:229-237 (1991). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

B. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression insoybean or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in soybean.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810-812 (1985) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genetics 231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3):291-300 (1992)).

The ALS promoter, Xba1/NcoI fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence similarity to said Xba1/NcoIfragment), represents a particularly useful constitutive promoter. SeePCT application W096/30530.

C. Tissue-specific or Tissue-preferred Promoters

A tissue-specific promoter is operably linked to a gene for expressionin soybean. Optionally, the tissue-specific promoter is operably linkedto a nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in soybean. Plants transformed with agene of interest operably linked to a tissue-specific promoter producethe protein product of the transgene exclusively, or preferentially, ina specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferredpromoter—such as that from the phaseolin gene (Murai et al., Science23:476482 (1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci.U.S.A. 82:3320-3324 (1985)); a leaf-specific and light-induced promotersuch as that from cab or rubisco (Simpson et al., EMBO J.4(11):2723-2729 (1985) and Timko et al., Nature 318:579-582 (1985)); ananther-specific promoter such as that from LAT52 (Twell et al., Mol.Gen. Genetics 217:240-245 (1989)); a pollen-specific promoter such asthat from Zm13 (Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993))or a microspore-preferred promoter such as that from apg (Twell et al.,Sex. Plant Reprod. 6:217-224 (1993).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondroin or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Close, P. S.,Master's Thesis, Iowa State University (1993), Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley”, Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol.91:124-129 (1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuokaet al., Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell.Biol. 108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon,et al., A short amino acid sequence able to specify nuclear location,Cell 39:499-509 (1984), Steifel, et al., Expression of a maize cell wallhydroxyproline-rich glycoprotein gene in early leaf and root vasculardifferentiation, Plant Cell 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is a soybean plant. In anotherpreferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR and SSR analysis, which identifies the approximate chromosomallocation of the integrated DNA molecule. For exemplary methodologies inthis regard, see Glick and Thompson, Methods in Plant Molecular Biologyand Biotechnology CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes That Confer Resistance to Pests or Disease and That Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the prodyuct of a corresponding avirulence (Avr)gene in the papthogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. Tomato encoddes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

B. A gene conferring resistance to a pest, such as soybean cystnematode. See e.g., PCT Application W096/30517; PCT ApplicationW093/19181.

C. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδendotoxin gene. Moreover, DNA molecules encoding δendotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va. forexample, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

D. A lectin. See, for example, the disclose by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

E. A vitamin-binding protein such as avidin. See PCT applicationUS93/06487, the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

F. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor 1), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor) and U. S. Pat. No.5,494,813 (Hepher and Atkinson, issued Feb. 27, 1996).

G. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

H. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

I. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

J. An enzyme responsible for a hyperaccumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

K. An enzyme involved in the modification, including theost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. BioL 23:691 (1993), who teach the nucleotide sequence ofa cDNA encoding tobacco hookworm chitinase, and Kawalleck et al., PlantMolec. Biol. 21:673 (1993), who provide the nucleotide sequence of theparsley ubi4-2 polyubiquitin gene.

L. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

M. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application W095/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

N. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-β, lytic peptide analog to rendertransgenic tobacco plants resistant to Pseudomonas solanacearum.

O. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaid\cvirus, tobacco streak virus, potato virus X, potato virus Y, tobaccoetch virus, tobacco rattle virus and tobacco mosaic virus. Id.

P. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

Q. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

R. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo α-1, 4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubartet al., Plant J. 2:367 (1992).

S. A development-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bioi/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivting gene havean increased resistance to fungal disease.

2. Genes That Confer Resistance to a Herbicide, For Example:

A. A herbicide that inhibits the growing point or meristem, such as animidazalinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance impaired by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase, PAT and Streptomyces hygroscopicusphosphinothricin-acetyl transferase, bar, genes), and pyridinoxy orphenoxy proprionic acids and cycloshexones (ACCase inhibitor-encodinggenes). See, for example, U.S. Pat. No.4,940,835 to Shah, et al., whichdiscloses the nucleotide sequence of a form of EPSP which can conferglyphosate resistance. A DNA molecule encoding a mutant aroA gene can beobtained under ATCC accession number 39256, and the nucleotide sequenceof the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai.European patent application No. 0 333 033 to Kumada et al., and U.S.Pat. No. 4,975,374 to Goodman et al., disclose nucleotide sequences ofglutamine synthetase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in Europeanapplication No. 0 242 246 to Leemans et al., DeGreef et al.,Bio/Technology 7:61 (1989), describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cycloshexones, such as sethoxydim andhaloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al., Theor. Appl. Genet. 83:435 (1992).

C. A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+genes) and a benzonitrile (nitrilase gene). Przibila et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

3. Genes That Confer or Contribute to a Value-Added Trait, Such as:

A. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearoyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci.U.S.A. 89:2624 (1992).

B. Decreased phytate content—1) Introduction of a phytase-encoding genewould enhance breakdown of phytate, adding more free phosphate to thetransformed plant. For example, see Van Hartingsveldt et al., Gene127:87 (1993), for a disclosure of the nucleotide sequence of anAspergillus niger phytase gene.

2) A gene could be introduced that reduced phytate content. In maize,this, for example, could be accomplished, by cloning and thenreintroducing DNA associated with the single allele which is responsiblefor maize mutants characterized by low levels of phytic acid. See Raboyet al., Maydica 35:383 (1990).

C. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteol. 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet. 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene), Penet al., Bio/Technology 10:292 (1992) (production of transgenic plantsthat express Bacillus lichenifonnis α-amylase), Elliot et al., PlantMolec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertasegenes), Søgaard et al., J. Biol. Chem. 268:22480 (1993) (site-directedmutagenesis of barley α-amylase gene), and Fisher et al., Plant Physiol.102:1045 (1993) (maize endosperm starch branching enzyme II).

Methods for Soybean Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

A. Agrobacterium-mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch et al., Science 227:1229 (1985). A. tumefaciens and A. rhizogenesare plant pathogenic soil bacteria which genetically transform plantcells. The Ti and Ri plasmids of A. tumefaciens and A, rhizogenes,respectively, carry genes responsible for genetic transformation of theplant. See, for example, Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991).Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are provided by Gruber et al.,supra, Miki et al., supra, and Moloney et al., Plant Cell Reports 8:238(1989). See also, U.S. Pat. No. 5,563,055 (Townsend and Thomas), issuedOct. 8, 1996.

B. Direct Gene Transfer

Several methods of plant transformation, collectively referred to asdirect gene transfer, have been developed as an alternative toAgrobacterium-mediated transformation. A generally applicable method ofplant transformation is microprojectile-mediated transformation whereinDNA is carried on the surface of microprojectiles measuring 1 to 4 μm.The expression vector is introduced into plant tissues with a biolisticdevice that accelerates the microprojectiles to speeds of 300 to 600 m/swhich is sufficient to penetrate plant cell walls and membranes. Sanfordet al., Part. Sci. Technol 5:27 (1987), Sanford, J. C., Trends Biotech.6:299 (1988), Klein et al., Bio/Technology 6:559-563 (1988), Sanford, J.C., Physiol Plant 7:206 (1990), Klein et al., Biotechnology 10:268(1992). See also U.S. Pat. No. 5,015,580 (Christou, et al.), issued May14, 1991; U.S. Pat. No. 5,322,783 (Tomes, et al), issued Jun. 21, 1994.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively,liposome or spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-omithine have also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Donn et al., In Abstracts of VIIth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990);D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spencer et al.,Plant Mol. Biol. 24:51-61 (1994).

Following transformation of soybean target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic variety. The transgenic variety could then becrossed, with another (non-transformed or transformed) variety, in orderto produce a new transgenic variety. Alternatively, a genetic traitwhich has been engineered into a particular soybean line using theforegoing transformation techniques could be moved into another lineusing traditional backcrossing techniques that are well known in theplant breeding arts. For example, a backcrossing approach could be usedto move an engineered trait from a public, non-elite variety into anelite variety, or from a variety containing a foreign gene in its genomeinto a variety or varieties which do not contain that gene. As usedherein, “crossing” can refer to a simple X by Y cross, or the process ofbackcrossing, depending on the context.

Tissue Culture of Soybeans

When the term soybean plant is used in the context of the presentinvention, this also includes any single gene conversions of thatvariety. The term single gene converted plant as used herein refers tothose soybean plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of a variety are recovered in additionto the single gene transferred into the variety via the backcrossingtechnique. Backcrossing methods can be used with the present inventionto improve or introduce a characteristic into the variety. The termbackcrossing as used herein refers to the repeated crossing of a hybridprogeny back to the recurrent parent. The parental soybean plant whichcontributes the gene for the desired characteristic is termed thenonrecurrent or donor parent. This terminology refers to the fact thatthe nonrecurrent parent is used one time in the backcross protocol andtherefore does not recur. The parental soybean plant to which the geneor genes from the nonrecurrent parent are transferred is known as therecurrent parent as it is used for several rounds in the backcrossingprotocol (Poehlman & Sleper, 1994; Fehr, 1987). In a typical backcrossprotocol, the original variety of interest (recurrent parent) is crossedto a second variety (nonrecurrent parent) that carries the single geneof interest to be transferred. The resulting progeny from this cross arethen crossed again to the recurrent parent and the process is repeateduntil a soybean plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred gene from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single gene of the recurrent variety ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphological,constitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross, one ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new variety but that can beimproved by backcrossing techniques. Single gene traits may or may notbe transgenic, examples of these traits include but are not limited to,male sterility, waxy starch, herbicide resistance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability andyield enhancement. These genes are generally inherited through thenucleus. Several of these single gene traits are described in U.S. Pat.Nos. 5,959,185, 5,973,234 and 5,977,445, the disclosures of which arespecifically hereby incorporated by reference.

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of soybeans andregeneration of plants therefrom is well know and widely published. Forexample, reference may be had to Komatsuda, T. et al., “Genotype XSucrose Interactions for Somatic Embryogenesis in Soybean,” Crop Sci.31:333-337 (1991); Stephens, P. A., et al., “Agronomic Evaluation ofTissue-Culture-Derived Soybean Plants,” Theor. Appl. Genet. (1991)82:633-635; Komatsuda, T. et al., “Maturation and Germination of SomaticEmbryos as Affected by Sucrose and Plant Growth Regulators in SoybeansGlycine gracilis Skvortz and Glycine max (L.) Merr.” Plant Cell, Tissueand Organ Culture, 28:103-113 (1992); Dhir, S. et al., “Regeneration ofFertile Plants from Protoplasts of Soybean (Glycine max L. Merr.);Genotypic Differences in Culture Response,” Plant Cell Reports (1992)11:285-289; Pandey, P. et al., “Plant Regeneration from Leaf andHypocotyl Explants of Glycine-wightii (W. and A.) VERDC. var.longicauda,” Japan J. Breed. 42:1-5 (1992); and Shetty, K., et al.,“Stimulation of In Vitro Shoot Organogenesis in Glycine max (Merrill.)by Allantoin and Amides,” Plant Science 81:245-251 (1992); as well asU.S. Pat. No. 5,024,944 issued Jun. 18, 1991 to Collins et al., and U.S.Pat. No. 5,008,200 issued Apr. 16, 1991 to Ranch et al., the disclosuresof which are hereby incorporated herein in their entirety by reference.Thus, another aspect of this invention is to provide cells which upongrowth and differentiation produce soybean plants having thephysiological and morphological characteristics of soybean variety9469069618964.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollen, flowers, seeds, pods, leaves,stems, roots, root tips, anthers, and the like. Means for preparing andmaintaining plant tissue culture are well known in the art. By way ofexample, a tissue culture comprising organs has been used to produceregenerated plants. U.S. Pat. Nos. 5,959,185; 5,973,234 and 5,977,445,described certain techniques, the disclosures of which are incorporatedherein by reference.

This invention also is directed to methods for producing a soybean plantby crossing a first parent soybean plant with a second parent soybeanplant wherein the first or second parent soybean plant is a soybeanplant of the variety 9469069618964. Further, both first and secondparent soybean plants can come from the soybean variety 9469069618964.Thus, any such methods using the soybean variety 9469069618964 are partof this invention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using soybean variety9469069618964 as a parent are within the scope of this invention,including those developed from varieties derived from soybean variety9469069618964. Advantageously, the soybean variety could be used incrosses with other, different, soybean plants to produce firstgeneration (F₁) soybean hybrid seeds and plants with superiorcharacteristics. The variety of the invention can also be used fortransformation where exogenous genes are introduced and expressed by thevariety of the invention. Genetic variants created either throughtraditional breeding methods using variety 9469069618964 or throughtransformation of 9469069618964 by any of a number of protocols known tothose of skill in the art are intended to be within the scope of thisinvention.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which soybean plants can be regenerated,plant calli, plant clumps, and plant cells that are intact in plants orparts of plants, such as embryos, pollen, ovules, flowers, pods, leaves,roots, root tips, anthers, and the like.

INDUSTRIAL USES

The seed of soybean variety 9469069618964, the plant produced from theseed, the hybrid soybean plant produced from the crossing of the varietywith any other soybean plant, hybrid seed, and various parts of thehybrid soybean plant can be utilized for human food, livestock feed, andas a raw material in industry.

The soybean is the world's leading source of vegetable oil and proteinmeal. The oil extracted from soybeans is used for cooking oil,margarine, and salad dressings. Soybean oil is composed of saturated,monounsaturated and polyunsaturated fatty acids. It has a typicalcomposition of 11% palmitic, 4% stearic, 25% oleic, 50% linoleic and 9%linolenic fatty acid content (“Economic Implications of Modified SoybeanTraits Summary Report”, Iowa Soybean Promotion Board and AmericanSoybean Association Special Report 92S, May 1990). Changes in fatty acidcomposition for improved oxidative stability and nutrition areconstantly sought after. Industrial uses of soybean oil which issubjected to further processing include ingredients for paints,plastics, fibers, detergents, cosmetics and lubricants. Soybean oil maybe split, interesterified, sulfurized, epoxidized, polymerized,ethoxylated, or cleaved. Designing and producing soybean oil derivativeswith improved functionality, oliochemistry, is a rapidly growing field.The typical mixture of triglycerides is usually split and separated intopure fatty acids, which are then combined with petroleum-derivedalcohols or acids, nitrogen, sulfonates, chlorine, or with fattyalcohols derived from fats and oils.

Soybean is also used as a food source for both animals and humans.Soybean is widely used as a source of protein for animal feeds forpoultry, swine and cattle. During processing of whole soybeans, thefibrous hull is removed and the oil is extracted. The remaining soybeanmeal is a combination of carbohydrates and approximately 50% protein.

For human consumption soybean meal is made into soybean flour which isprocessed to protein concentrates used for meat extenders or specialtypet foods. Production of edible protein ingredients from soybean offersa healthy, less expensive replacement for animal protein in meats aswell as dairy-type products.

TABLES

In Tables 1-3 that follow, the traits and characteristics of soybeancultivar 9469069618964 are compared to several competing varieties ofcommercial soybeans of similar maturity. The first three lines of thetable indicate the overall mean, number of locations and LSD amounts foreach category tested. Column 1 shows the variety tested; column 2 showsthe yield in bushels/acre (BU/A) for the instant invention and thecompetitor varieties. Column 3 indicates the days to maturity (MAT)after August 31. Column 4 shows the plant height (HGT) in inches for theinstant invention and the competitor varieties. Column 5 indicates theplant lodging (LDG). Column 6 shows the general appearance rating (GR)scores for the instant invention and the competitor varieties and column7 shows emergence (EMR) ratings. Lodging, General Appearance andEmergence Rating scores are rated 1=Best and 9=Worst.

TABLE 1 1997 AGRONOMIC DATA BU/A MAT HGT LDG GR EMR Overall Mean 53.7915.70 38.30 2.20 2.50 2.40 # of Locations 4 4 3 3 3 3 LSD 946906961896455.80 9.30 38.00 1.50 2.50 1.00 Asgrow Al923 68.89 19.00 36.00 1.00 1.003.00 Asgrow AG2101 65.13 20.00 36.50 1.00 1.00 3.00 Asgrow AG1901 59.0018.30 43.00 2.50 2.50 2.00 Asgrow A1553 58.14 15.30 34.50 2.00 2.00 3.00

TABLE 2 1998 AGRONOMIC DATA BU/A MAT HGT LDG GR EMR Overall Mean 56.8712.70 33.60 1.20 1.90 1.80 # of Locations 11 11 11 11 11 7 LSD 2.41 1.791.13 0.28 0.31 0.45 9469069618964 57.24 12.20 36.10 1.10 1.50 2.00Asgrow A1553 63.43 18.00 33.30 1.10 1.40 1.80 Asgrow A0868 63.05 13.9032.70 1.20 1.40 1.70 Bayfield 54.34 9.00 31.00 1.20 2.40 1.60 NK S09-9556.18 12.20 35.70 1.30 2.00 1.30 Asgrow AG1101 57.34 13.80 38.80 1.702.00 1.50 Asgrow AG0901 54.69 12.70 35.60 1.50 1.80 1.00 Asgrow A094953.17 12.60 36.20 1.40 2.10 1.50

TABLE 3 1999 AGRONOMIC DATA BU/A MAT HGT LDG GR EMR Overall Mean 54.9016.50 31.90 2.20 2.70 2.60 # of Locations 15 15 15 15 15 15 LSD 3.121.62 1.28 0.73 0.94 1.55 9469069618964 56.20 19.80 35.30 1.90 2.00 2.50Asgrow A1101 56.10 15.10 32.00 2.40 2.10 3.50 Asgrow AG0901 54.01 15.3034.50 2.80 3.50 2.70 Dekalb CX085RR 54.40 17.10 28.80 1.50 3.50 2.70Dekalb CX105RR 54.00 22.10 36.80 2.60 4.00 2.70 Dekalb CX099RR 52.4015.10 31.30 2.30 2.00 2.20

DEPOSIT INFORMATION

A deposit of the Asgrow Seed Company LLC proprietary soybean cultivar9469069618964 disclosed above and recited in the appended claims hasbeen made with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110. The date of deposit was May14, 2002. The deposit of 2,500 seeds were taken from the same depositmaintained by Asgrow Seed Company LLC since prior to the filling date ofthis application. All restrictions upon the deposit have been removed,and the deposit is intended to meet all of the requirements of 37 C.F.R.§1.801-1.809. The ATCC accession number is PTA-4347. The deposit will bemaintained in the depository for a period of 30 years, or 5 years afterthe last request, or for the effective life of the patent, whichever islonger, and will be replaced as necessary during that period.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding. However, it will be obvious that certain changes andmodifications such as single gene modifications and mutations,somoclonal variants, variant individuals selected from large populationsof the plants of the instant variety and the like may be practicedwithin the scope of the invention, as limited only by the scope of theappended claims.

What is claimed is:
 1. Seed of a soybean variety designated9469069618964, representative seed having been deposited under ATCCAccession No. PTA-4347.
 2. A soybean plant, or parts thereof, producedby growing the seed of claim
 1. 3. Pollen of the plant of claim
 2. 4. Anovule of the plant of claim
 2. 5. A tissue culture of regenerable cellsfrom the plant of claim 2, representative seed having been depositedunder ATCC Accession No. PTA-4347.
 6. A tissue culture according toclaim 5, the cells or protoplasts of the tissue culture being from atissue selected from the group consisting of: leaves, pollen, embryos,cotyledon, hypocotyl, meristematic cells, roots, root tips, anthers,flowers, seeds, stems and pods.
 7. A soybean plant regenerated from thetissue culture of claim 5, wherein the regenerated plant is capable ofexpressing all of the morphological and physiological characteristics ofsoybean cultivar
 9469069618964. 8. A method for producing a hybridsoybean seed comprising crossing a first parent soybean plant with asecond parent soybean plant and harvesting the resultant hybrid soybeanseed, wherein said first parent soybean plant or said second parentsoybean plant is the soybean plant of claim
 2. 9. A hybrid soybean seedproduced by the method of claim
 8. 10. A hybrid soybean plant, or partsthereof, produced by growing said hybrid soybean seed of claim
 9. 11. Amethod for producing a soybean variety 9469069618964-derived soybeanplant, comprising: a) crossing soybean variety 9469069618964,representative samples of said variety having been deposited under ATCCaccession number , with a second soybean plant to yield progeny soybeanseed; and b) growing said progeny soybean seed, under plant growthconditions, to yield said soybean variety 9469069618964-derived soybeanplant.
 12. The method of claim 11, further comprising: c) crossing saidsoybean variety 9469069618964-derived soybean plant with itself oranother soybean plant to yield additional soybean variety9469069618964-derived progeny soybean seed; d) growing said progenysoybean seed of step (a) under plant growth conditions, to yieldadditional soybean variety 9469069618964-derived soybean plants; and e)repeating the crossing and growing steps of (a) and (b) from 0 to 7times to generate further soybean variety 9469069618964-derived soybeanplants.
 13. The soybean plant, or parts thereof, of claim 5,representative seed having been deposited under ATCC Accession No.PTA-4347, wherein the plant or parts thereof have been transformed sothat its genetic material contains one or more transgenes operablylinked to one or more regulatory elements.
 14. A method for producing asoybean plant that contains in its genetic material one or moretransgenes, comprising crossing the soybean plant of claim 13, witheither a second plant of another soybean variety, or a non-transformedsoybean plant of the soybean variety 9469069618964, so that the geneticmaterial of the progeny that result from the cross contains thetransgene(s) operably linked to a regulatory element.
 15. Soybeanplants, or parts thereof, produced by the method of claim 14.